The present invention relates to novel compounds that are capable of covalently modifying covalently modifying xcex2-tubulin and acting as therapeutic and diagnostic agents, and acting as therapeutic and diagnostic agents.
Microtubules are subcellular organelles located in most eukaryotic cells and are involved in a variety of cell functions including mitosis, intracellular movement, cell movement and maintenance of cell shape. Microtubule assembly involves polymerization of tubulin and additional construction with other components of the microtubule (referred to as xe2x80x9cmicrotubule-associated proteinsxe2x80x9d or MAPs).
Tubulin itself consists of two 50 kDa subunits Tubulin itself consists of two 50 kDa subunits (xcex1- and xcex2-tubulin) which combine in a heterodimer. The heterodimer binds two molecules of guanosine triphosphate (GTP). One of the GTP molecules is tightly bound and cannot be removed without denaturing the heterodimer, while the other GTP molecule is freely exchangeable with other GTPs. This exchangeable GTP is believed to be involved in tubulin function. In particular, the tubulin heterodimer can combine in a head-to-tail arrangement in the presence of GTP to form a long protein fiber, known as a protofilament. These protofilaments can then group together to form a protein sheet which then curls into a tube-like structure known as a microtubule. Interference with this process of microtubule construction affects the downstream processes of mitosis and maintenance of cell shape.
Most of the naturally-occurring antimitotic agents have been shown to exert their effect by binding to tubulin, rather than MAPs or other proteins involved in mitosis. For example, tubulin is the biochemical target for several clinically useful anticancer drugs, including vincristine, vinblastine and paclitaxel. Another natural product, colchicine, was instrumental in the purification of tubulin as a result of its potent binding, with xcex2-tubulin being the target for colchicine. Colchicine and other colchicine site agents bind at a site on xcex2-tubulin that results in inhibition of a cross-link between cys-239 and cys-354 (wherein the numbering refers to the between cys-239 and cys-354 (wherein the numbering refers to the xcex22 isotype) by such non-specific divalent sulfhydryl reactive agents as N,Nxe2x80x2-ethylenebis-iodoacetamide. However, simple alkylation of cys-239 does not appear to inhibit colchicine binding to tubulin.
In addition to colchicine, other natural products are known that bind at the colchicine site and inhibit microtubule assembly, for example, podophyllotoxin, steganacin and combretastatin. Still other agents bind to sites on tubulin referred to as the Vinca alkaloid site and the Rhizoxin/Maytansine site. However, none of the noted natural products are thought to operate by covalent modification of tubulin.
The present invention provides natural product derivatives as well as derivatives of known tubulin-binding compounds in which a (poly)fluorobenzene, a fluoropyridine, or a fluoronitrophenyl moiety is incorporated or added to the structure. These derivatives can be used as antimitotic agents and can be considered covalent modifiers of tubulin. The strategy developed for each of the compounds is to i) append a fluorinated electrophile (e.g., pentafluorophenylsulfonamido, 2-fluoropyridyl, or 3,5-dinitro-4-fluorophenyl) to an existing functional group in a natural product, ii) replace an aromatic ring in a natural product with a fluorinated electrophile, or iii) attach a fluorinated electrophile to an open valence in a portion of the molecule that will not interfere with recognition and binding to the tubulin site. Derivatives are provided based on colchicine, steganacin, podophyllotoxin, nocodazole, combretastatin, curacin A, vinblastine, vincristine, dolastatin, 2-methoxyestradiol, dihydroxy-pentamethoxyflavanone and others.
The present invention further provides pharmaceutical compositions containing the natural product derivatives as well as therapeutic and diagnostic methods using those compounds and compositions.
Definitions
The term xe2x80x9cpharmaceutically acceptable saltsxe2x80x9d is meant to include salts of the active compounds which are prepared with relatively nontoxic acids or bases, depending on the particular substituents found on the compounds described herein. When compounds of the present invention contain relatively acidic functionalities, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salt, or a similar salt. When compounds of the present invention contain relatively basic functionalities, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the salts derived from relatively nontoxic organic acids like acetic, propionic, isobutyric, oxalic, maleic, malonic, benzoic, succinic, suberic, fumaric, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Also included are salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galactunoric acids and the like (see, for example, Berge, S. M., et al, xe2x80x9cPharmaceutical Saltsxe2x80x9d, Journal of Pharmaceutical Science, 1977, 66, 1-19). Certain specific compounds of the present invention contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts.
The neutral forms of the compounds may be regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salts are equivalent to the parent form of the compound for the purposes of the present invention.
In addition to salt forms, the present invention provides compounds which are in a prodrug form. Prodrugs of the compounds described herein are those compounds that readily undergo chemical changes under physiological conditions to provide a compound of formula I. Additionally, prodrugs can be converted to the compounds of the present invention by chemical or biochemical methods in an ex vivo environment. For example, prodrugs can be slowly converted to the compounds of the present invention when placed in a transdermal patch reservoir with a suitable enzyme.
Certain compounds of the present invention can exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are intended to be encompassed within the scope of the present invention. Certain compounds of the present invention may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present invention and are intended to be within the scope of the present invention.
Certain compounds of the present invention possess asymmetric carbon atoms (optical centers) or double bonds; the racemates, diastereomers, geometric isomers and individual isomers are all intended to be encompassed within the scope of the present invention.
The compounds of the present invention may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, the compounds may be radiolabeled with radioactive isotopes, such as for example tritium (3H), iodine-125 (125I) or carbon-14 (14C). All isotopic variations of the compounds of the present invention, whether radioactive or not, are intended to be encompassed within the scope of the present invention.
General
The present invention provides a variety of agents capable of covalent attachment to tubulin. Accordingly, the compounds are particularly useful as antimitotic agents for the treatment of cancer. The compounds are derivatives of naturally-occurring antimitotic agents as well as other tubulin-interacting compounds. Briefly, the compounds can be described as antimitotic agents having, for example, a pentafluorophenyl-sulfonamide group (C6F5xe2x80x94SO2xe2x80x94NHxe2x80x94), a 2-fluoropyridyl group, a nitrofluorophenyl group or a dinitrofluorophenyl group. In each instance, the reactive fluorinated aromatic moiety is introduced into the parent compound by replacing an existing portion of the parent (e.g., an aromatic ring or lactone), by attaching to an available reactive functional group (e.g., hydroxyl, amino, carboxylic acid and the like), or by attaching to an otherwise unfunctionalized portion of the molecule. Each of the reactive fluorinated aromatic moieties is capable of covalently modifying a cysteine thiol owing to the electrophilic nature of the fluoroaryl moiety and the leaving group character of the fluorine atom.
Derivatives of parent tubulin-interacting compounds are also described in which small portions of the parent compound are replaced with fragments of similar size that can increase the reactivity of the aromatic electrophile. For example, an ethylene group (xe2x80x94CH2CH2xe2x80x94) can be replaced with a sulfonamido moiety (xe2x80x94SO2NHxe2x80x94) in those positions wherein the reactivity of an adjacent pentafluorophenyl or tetrafluorophenyl group can be enhanced. Additionally, any of the noted fluorinated romatic electrophiles can be attached to the remainder of the molecule via a connecting element that further enhances the reactivity of the fluorinated electrophile (e.g., a sulfonyl group or a carbonyl group).
The tubulin-interacting agents on which the following embodiments are based have been described in, for example, Jordan, et al., Med. Res. Rev. 18(4):259-296 (1998), Bai, et al., J Biol. Chem. 271(21):12639-12645 (1996), Hamel, Med. Res. Rev. 16(2):207-231 (1996), Sackett, Pharmacol. Ther. 59(2):163-228 (1993) and Luduena, et al., Pharmac. Ther. 49:133-152 (1991).
The present invention generally provides tubulin binding agents that selectively and covalently bind to tubulin. The agents are derivatives of compounds which non-covalently bind to the colchicine binding site, the vinca alkaloid binding site, or the rhizoxin/maytansine binding site of tubulin. Additionally, the derivatives are formed by the attachment of a fluorinated aromatic electrophile to the parent non-covalent compounds, or by the replacement of a portion of the parent compound with the fluorinated aromatic electrophile. As used herein, the term derivative is also meant to include those agents in which a fluorinated aromatic electrophile is attached to the parent compound via a linker, preferably a linker which increases the electrophilic character of the fluorinated aromatic electrophile. Still further, the term xe2x80x9cderivativexe2x80x9d is meant to include those compounds in which small portions of the parent compound are replaced with fragments of similar size that also serve to enhance the reactivity of the fluorinated aromatic electrophile.
In preferred embodiments, the fluorinated aromatic electrophile comprises a pentafluorophenyl, tetrafluorophenyl, 2-fluoropyridyl, dinitrofluorophenyl or fluoronitrophenyl group.
In other preferred embodiments, the agent is a derivative of a compound selected from the group consisting of colchicine, podophyllotoxin, combretastatin, nocodazole, stegnacin, dihydroxy-pentamethoxyflananone, 2-methoxyestradiol, vinblastine, vincristine, dolastatin, curacin A, etoposide, teniposide, sanguinarine, griseofulvin, cryptophycins or chelidonine.
The invention is better understood with reference to the following non-limiting embodiments.
Colchicine Derivatives
Colchicine (i) binds to tubulin at a site, now termed xe2x80x9cthe colchicine binding site,xe2x80x9d proximately located near Cys-239 of tubulin. Although colchicine has been referred to as an irreversible inhibitor of tubulin assembly, the precise mechanism of its action is unknown. Inspection of colchicine""s chemical structure does not suggest that it operates via covalent attachment to tubulin. In an effort to develop more effective inhibitors of tubulin assembly, the present invention provides derivatives of colchicine that are capable of covalently binding to nucleophilic residues in the colchicine binding site (e.g., Cys-239 or other cysteine thiol groups). 
One group of embodiments is shown as i-a, in which the xe2x80x9cAxe2x80x9d ring of colchicine (the trimethoxybenzo group) has been replaced with a 2-fluoropyrido group. The 2-fluoropyrido group is known to be activated toward nucleophiles at the 2-position, ultimately resulting in displacement of the fluoro substituent. While i-a shows a 4,5-fused 2-fluoropyrido moiety, one of skill in the art will understand that ring fusion could also be at the 3,4 carbon bond or the 5,6 carbon bond and in either orientation. See, for example, the embodiments shown below as i-a1 through i-a5. 
Another fused-ring replacement is shown above as i-c. In this group of embodiments, the xe2x80x9cAxe2x80x9d ring of colchicine has been replaced with a dinitrofluorobenzo group which also gives rise to multiple embodiments. For example, the dinitrofluorobenzo moiety can be fused to the xe2x80x9cBxe2x80x9d ring of colchicine (the cycloheptane ring) using either of two positional orientations to give rise to two compounds (shown as i-c and i-c3). Additionally, the placement of the nitro groups can be altered such that they occupy positions ortho and para to the fluorine atom. In this orientation, the electron-withdrawing character of the nitro groups is very similar to the character imparted when they occupy the two positions which are ortho to the fluorine atom (see i-c1 and i-c2). 
Additional embodiments involve the modification of functional groups already present on colchicine. For example, the acetamido group present in colchicine can be modified to contain a moiety capable of covalently attaching to a colchicine binding site nucleophile (see i-b). The pentafluorophenylsulfonyl group has been shown to be suitable for covalent attachment to Cys-239 of tubulin (see, WO 98/05315). In addition to the embodiment illustrated as i-b, the present invention provides other positional isomers in which the colchicine acetamido group is removed and a pentafluorophenylsulfonamido group is attached at any of the available valences (five additional valences) of the cycloheptane ring.
Still other embodiments are provided in which one of the methoxy groups of the colchicine xe2x80x9cAxe2x80x9d ring is replaced with a pentafluorophenylsulfonamido group (see, for example, i-d). One of skill in the art will understand that any of the methoxy groups can be replaced with the pentafluorophenylsulfonamido group (or an equivalent moiety for covalent attachment to tubulin). Still further, a pentafluorophenylsulfonamido group can be attached to the remaining open valence on the colchicine xe2x80x9cAxe2x80x9d ring, preferably with removal of the adjacent methoxy group.
Podophyllotoxin
Podophyllotoxin (ii) is an antimitotic agent that was first isolated from plants about 100 years ago, and more recently has been shown to exert its effects by binding to tubulin at the colchicine binding site. Interestingly, a computer modeling study suggests that there is an incomplete overlap between the colchicine binding site and the site occupied by podophyllotoxin, with the two trimethoxybenzene rings not binding in equivalent sites. Additionally, podophyllotoxin binding to tubulin is more rapid and reversible than the binding of colchicine. New derivatives are provided in the present invention which are designed to correct the deficiency of reversible binding. 
In a first group of embodiments, the podophyllotoxin xe2x80x9cExe2x80x9d ring (the trimethoxyphenyl ring) is removed and a pentafluorophenylsulfonamido group is attached to the xe2x80x9cBxe2x80x9d ring (see ii-a) to provide an electrophilic fluorinated aromatic ring in the approximate position of the removed xe2x80x9cExe2x80x9d ring.
In other embodiments, the xe2x80x9cExe2x80x9d ring is replaced with either a dinitrofluorophenyl moiety (e.g., ii-b) or a 2-fluoropyridyl moiety (e.g., ii-c). Positional isomers of ii-b and ii-c are also contemplated by the present invention. Thus, for either group of embodiments, attachment of the electrophilic aromatic ring can be through any available valence. Preferably, attachment is through a position that renders the fluorine most available for displacement by a binding site nucleophile.
In still other embodiments, the podophyllotoxin hydroxyl group (attached to the xe2x80x9cCxe2x80x9d ring) is replaced by a group suitable for covalent attachment to tubulin (e.g., a pentafluorophenylsulfonamido group, see ii-d).
Combretastatin
The combretastatins are another group of plant-derived antimitotic agents. First isolated and characterized in the early 1980""s, the combretastatins (of which combretastatin A-4 (iii) is one of the most potent members) have been shown to bind to tubulin in a competitive manner to colchicine. Additional evidence has shown that the combretastatins bind rapidly and reversibly to tubulin. 
Incorporation of a covalent binding moiety into the combretastatin molecule is illustrated with compounds iii-a through iii-d. Compounds iii-a and iii-b exemplify the approach in which the trimethoxyphenyl (or xe2x80x9cAxe2x80x9d) ring of combretastatin A-4 is replaced with a fluorinated electrophile group such as 2-fluoropyridyl and 3,5-dinitro-4-fluorophenyl. In each case, the point of attachment to the remainder of the molecule can be at the indicated position or at any other open valence of the fluorinated aromatic ring.
Similarly, the xe2x80x9cAxe2x80x9d ring of combretastatin can be replaced with a pentafluorophenyl group as indicated in iii-c. To further enhance the electrophilicity of the fluorinated aromatic ring, a sulfonamide bridging group (xe2x80x94SO2NHxe2x80x94) is added to effectively form a vinylous pentafluorophenyl sulfonamido group.
In yet another group of embodiments, one of the methoxy groups of the combretastatin xe2x80x9cAxe2x80x9d ring can be replaced with a pentafluorophenylsulfonamido group (see, for example, iii-d). One of skill in the art will understand that any of the methoxy groups can be replaced with the pentafluorophenylsulfonamido group (or an equivalent moiety for covalent attachment to tubulin). Still further, a pentafluorophenylsulfonamido group can be attached to the remaining open valence on the combretastatin xe2x80x9cAxe2x80x9d ring, preferably with removal of the adjacent methoxy group.
The above modifications to combretastatin have been designed, developed and are illustrated with reference to the xe2x80x9cAxe2x80x9d ring of combretastatin. The present invention, however, is not so limited and provides additional embodiments in which similar modifications are carried out on the combretastatin xe2x80x9cBxe2x80x9d ring.
Nocodazole
Nocodazole (iv) was discovered as part of an antiparasitic program. Nocodazole and related benzimidazole derivatives have been shown to be competitive inhibitors of colchicine binding to tubulin. Some have postulated that the phenyl ring of iv binds to the same pocket of tubulin as the trimethoxyphenyl ring of colchincine. 
In one group of embodiments, the thienyl moiety of nocodazole is replaced with an electrophilic fluorinated aryl or heteroaryl moiety (see, for example, iv-a and iv-d). As with the other embodiments using fluorinated aryl or heteroaryl moieties, the point of attachment to the remainder of the molecule can be at any available valence on the aromatic ring. Additionally, the aryl group shown in iv-a as a 3,5-dinitro-4-fluorophenyl group can be substituted with the electronically similar 3,5-dinitro-2-fluorophenyl group and its equivalents (see, for example, isomers iv-a1 through iv-a4). 
In another group of embodiments, the methyl carbamate present on the benzimidazole portion of nocodazole is replaced with a pentafluorophenylsulfonamido moiety (see iv-b). One of skill in the art will appreciate that other electrophilic aryl groups could be substituted for the pentafluorophenyl group, for example, a 3-nitro-4-fluorophenyl group.
In still other embodiments, nocodazole is modified to include an electrophilic fluorinated aryl or heteroaryl moiety at an open valence on the parent molecule (see, for example, iv-c).
2-Methoxyestradiol
In yet another group of embodiments, compounds are provided that are related to 2-methoxyestradiol (v). Recently, 2-methoxyestradiol has been shown to be a weak competitive inhibitor of the binding of colchicine to tubulin and also inhibits the rate, but not the extent of tubulin assembly. Additional studies showed that rapid binding of 2-methoxyestradiol to unpolymerized tubulin could be inhibited with colchicine and other colchicine site agents. Still further work indicated that 2-methoxyestradiol binds to polymerized tubulin and that the altered properties of polymer formed in the presence of the agent may be due to this post-polymerization binding. Regardless of the precise mechanism of action, the compounds provided herein are capable of covalently modifying either tubulin itself or polymerized tubulin to modulate microtubule assembly. 
Application of the general design strategies, provided in other embodiments above, to 2-methoxyestradiol provides, for example, compounds v-a through v-d. In compound v-a, the aromatic ring of 2-methoxyestradiol is replaced with a dinitrofluorobenzene ring. Similarly, a 2-fluoropyridine ring could be substituted for the dinitrofluorobenzene ring to form additional compounds. For each of these two groups of fused-ring substitutions, positional embodiments similar to those described for i-a and i-c are also within the scope of the present invention.
Further embodiments are exemplified by compound v-b. As shown, compound v-b contains the entire steroid structure of 2-methoxyestradiol with an appended pentafluorophenylsulfonamido group attached at a position adjacent to the xe2x80x9cDxe2x80x9d ring hydroxy group. Attachment can also be carried out at any other position which can be readily functionalized by well-known methods in the steroid literature.
Addition of a 2-fluoropyridyl moiety to the estradiol nucleus constitutes yet another embodiment of the present invention (see, for example, v-c).
Finally, replacement of the aromatic ring and two xe2x80x9cBxe2x80x9d ring carbon atoms with a tetrafluorophenylsulfonamido moiety results in v-d.
Dihydroxy-pentamethoxyflavanone
Flavonol 2 (vi) is yet another compound isolated from higher plants which has been shown to be moderately cytotoxic and inhibit tubulin polymerization as well as the binding of colchicine to tubulin. 
Compounds vi-a through vi-c illustrate that portion of the present invention in which the fused trimethoxyphenol (the xe2x80x9cAxe2x80x9d ring) is replaced with a tetrafluorobenzo group (vi-a), a 2-fluoropyrido group (vi-b) or a fluoronitrobenzo group (vi-c). In the case of the latter two compounds, positional isomers in which ring fusion occurs at other linkages on the electrophilic aromatic ring are also part of the present invention. In still other embodiments, the present invention provides those compounds and compositions in which the guaiacol group is replaced by a pentafluorophenyl, 2-fluoropyridyl, or a nitrofluorophenyl substituent.
Steganacin
Steganacin (vii) and a number of related compounds were initially isolated from the stems and stem bark of the East African tree Steganotaenia araliacea in the early 1970s. Steganacin shows significant similarity to both colchicine and podophyllotoxin. Accordingly, the strategies used to develop compounds derived from each of those natural products can be similarly applied to steganacin. 
In a first group of embodiments, the trimethoxybenzo moiety of steganacin is replaced with a fluoronitrobenzo group. Compound vii-a shows one isomeric embodiment. The present invention also contemplates those embodiments in which the fused 1,3-benzodioxole group is similarly replaced with a 2-fluoropyrido or nitrofluorobenzo group.
In another group of embodiments, the trimethoxybenzo moiety of steganacin is replaced with a tetrafluorobenzo group, while two carbons of the cyclooctane ring are replace with a sulfonamide linkage to provide additional activation toward nucleophilic substitution on the fluorinated aromatic ring (see vii-b).
In yet another group of embodiments, the reactive lactone of steganacin is replaced by a covalent attaching group (e.g., 2-fluoropyridyl) to provide compounds such as vii-c
In still other embodiments, a functional group can be removed and replaced with a covalent attaching group as exemplified in vii-d. Here, a methoxy group on the trimethoxybenzo portion of steganacin has been replaced by a pentafluorophenylsulfonamido moiety.
2-Phenyl-4-Quinolones
2-Phenyl-4-quinolones (exemplified by 6-(1-pyrolidinyl)-2-(3-methoxyphenyl)-4-quinolone (viii)) have been shown to be potent inhibitors of tubulin polymerization, and can arrest cell growth in a number of human tumor cell lines. 
In one group of embodiments, the xe2x80x9cAxe2x80x9d ring of viii is replaced with a fluoropyridine ring (see viii-a). In other embodiments, an electrophilic fluorinated aryl moiety (e.g., pentafluorophenylsulfonyl) is attached to the quinolone nitrogen or to a nitrogen atom attached to the 6-position of the quinolone. In still other embodiments, the pyrolidinyl moiety of viii is replaced with, for example, dinitrofluorophenyl (see, viii-d).
ER-34410
ER-34410 (ix) is an antitumor agent first described in WO 95/03279. The compound was shown to have an IC50 of 0.11 (g/mL against KB cells (human nasal cavity cancer) and is thought to bind noncovalently to the colchicine-binding site of tubulin. 
In one group of embodiments, the anisole ring of ER-34410 is removed and replaced with an electrophilic fluorinated aryl or heteroaryl group such as, for example, a 2-fluoropyridyl group (ix-a, shown attached through the 5-position of the pyridine ring), or a fluoronitrophenyl group (ix-b, shown attached through a position para to the fluorine atom).
In other embodiments, the entire 4-methoxyphenylsulfonamido group of ix can be removed and a pentafluorophenylsulfonyl group can be attached to either of the remaining nitrogens (those present in the 7-membered ring, to provide, for example, ix-c).
Vinblastine
Vinblastine (x), one of the Vinca alkaloids, has been used in the treatment of neoplastic diseases for several decades. While its antimitotic properties have been known almost since its discovery, the precise mechanism by which vinblastine achieves such disruption is less well understood. 
In one group of embodiments, the acetate functional group of vinblastine (x) is replaced by, for example, a pentafluorophenylsulfonamido group (see, x-a). In other embodiments, a pentafluorophenylsulfonyl group (or similar electrophilic moiety) is attached to the secondary indole nitrogen atom (see, x-b).
General Synthesis
In general, the compounds provided herein can be prepared using well-developed methodology for manipulation of functional groups on the parent compounds, or by modifications of synthetic methods used in the total synthesis of the parent natural products. For example, deacetylcolchicine (described by Lebeau, et al., Synth. Commun. 27:293-296 (1997)) can be sulfonylated with commercially available agents such as pentafluorophenylsulfonylchloride to produce i-b. Other synthesis methods are provided in the Examples.
Analysis of compounds
The compounds and compositions of the present invention exert their cytotoxic effects by interacting with cellular tubulin in a manner that is believed to be covalent and irreversible. Compounds and compositions may be evaluated in vitro for their ability to inhibit cell growth, for example, as described in Ahmed et al. (J Immunol. Methods 1994, 170, 211). Established animal models to evaluate antiproliferative effects of compounds are also known in the art. For example, compounds can be evaluated for their ability to inhibit the growth of human tumors grafted into immunodeficient mice using methodology similar to that described by Rygaard and Povlsen (Acta Pathol. Microbiol. Scand. 1969, 77, 758) and Giovanella and Fogh (Adv. Cancer Res. 1985, 44, 69).
Formulation and Administration of Compounds and Pharmaceutical Compositions
The invention provides methods of using the subject compounds and compositions to treat disease or provide medicinal prophylaxis by slowing and/or reducing the growth of tumors, etc. These methods generally involve contacting the cell with or administering to the host an effective amount of the subject compounds or pharmaceutically acceptable compositions. The compositions and compounds of the invention and the pharmaceutically acceptable salts thereof can be administered in any effective way such as via oral, parenteral or topical routes. Generally, the compounds are administered in dosages ranging from about 2 mg up to about 2,000 mg per day, although variations will necessarily occur depending on the disease target, the patient, and the route of administration. Preferred dosages are administered orally in the range of about 0.05 mg/kg to about 20 mg/kg, more preferably in the range of about 0.05 mg/kg to about 2 mg/kg, most preferably in the range of about 0.05 mg/kg to about 0.2 mg per kg of body weight per day.
In one embodiment, the invention provides the subject compounds combined with a pharmaceutically acceptable excipient such as sterile saline or other medium, water, gelatin, an oil, etc. to form pharmaceutically acceptable compositions. The compositions and/or compounds may be administered alone or in combination with any convenient carrier, diluent, etc. and such administration may be provided in single or multiple dosages. Useful carriers include solid, semi-solid or liquid media including water and non-toxic organic solvents.
In another embodiment, the invention provides the subject compounds in the form of a pro-drug, which can be metabolically converted to the subject compound by the recipient host. A wide variety of pro-drug formulations are known in the art.
The compositions may be provided in any convenient form including tablets, capsules, lozenges, troches, hard candies, powders, sprays, creams, suppositories, etc. As such the compositions, in pharmaceutically acceptable dosage units or in bulk, may be incorporated into a wide variety of containers. For example, dosage units may be included in a variety of containers including capsules, pills, etc.
The compositions may be advantageously combined and/or used in combination with other antiproliferative therapeutic agents, different from the subject compounds. In many instances, administration in conjunction with the subject compositions will enhance the efficacy of such agents. Exemplary antiproliferative agents include cyclophosphamide, methotrexate, adriamycin, cisplatin, daunomycin, vincristine, vinblastine, vinarelbine, paclitaxel, docetaxel, tamoxifen, flutamide, hydroxyurea, and mixtures thereof.
The compounds and compositions also find use in a variety of in vitro and in vivo assays, including diagnostic assays. In certain assays and in in vivo distribution studies, it is desirable to used labeled versions of the subject compounds and compositions, e.g. radioligand displacement assays. Accordingly, the invention provides the subject compounds and compositions comprising a detectable label, which may be spectroscopic (e.g. fluorescent), radioactive, etc.
The following examples provide more detailed descriptions of synthetic methods used to prepare compounds of the present invention. One of skill in the art will appreciate that many of the methods provided below are applicable to the modification or derivatization of other known noncovalent tubulin-binding agents. Accordingly, the examples are offered by way of illustration and not by way of limitation.